Materials and methods for drug delivery and uptake

ABSTRACT

The subject invention pertains to novel materials and methods for use in delivering and sequestering substances, such as pharmacological agents, within a patient. One aspect of the invention is directed towards core-shell particles having a core encapsulated within a calcium carbonate shell, with an intermediate layer composed of an amphiphilic compound surrounding the core. When the particles of the subject invention are administered to a patient, they are capable of removing lipophilic drugs by absorption of the drug through their mineral shell and into their core. The particles of the subject invention can also be administered to a patient as controlled release, drug delivery vehicles. Thus, in another aspect, the subject invention concerns a method of delivering pharmacological agents by administering the core-shell particles of the subject invention to a patient in need of such administration.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.10/243,340, filed Sep. 13, 2002, which is hereby incorporated byreference herein in its entirety, including any figures, tables, nucleicacid sequences, amino acid sequences, and drawings.

GOVERNMENT SUPPORT

The subject invention was made with government support under a researchproject supported by National Science Foundation Grant No. EEC-9402989.The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Treatment of drug overdose in humans, whether due to therapeuticmiscalculation, illicit drug use, or suicide attempt, presents a majorproblem to the health care industry worldwide. In the United Statesalone, over 300,000 patients are admitted to the emergency rooms becauseof drug overdose. Treatment of these patients costs the healthcareindustry over ten billion dollars because of hospital expenses and lostemployee productivity. This does not include the $80 billion associatedwith alcohol abuse (Moudgil, B. M., Seventh Year Annual Report. 2001:Engineering Research Center for Particle Science and Technology,University of Florida).

Current treatment protocols for overdosed patients vary with the drug ofconcern, but are focused on three objectives: prevention of drugabsorption, enhancement of drug excretion, and administration ofpharmacological antidotes. The first two are accomplished withtechniques nonspecific to the ingested drug, such as emesis, gastriclavage, or use of activated charcoal for the former objective, anddialysis or hemoperfusion for the latter. However, since absorption oftoxic drugs is very time sensitive, and since these techniques areapplied only once a patient reaches the emergency room, they are not aseffective as would be desired, with some techniques reported to recoveronly 30% of the ingested drug (Rumack, B. H., Poisoning: Prevention ofabsorption, in Poisoning and Overdose, M. J. Bayer and B. H. Rumack,Eds., 1983, p. 13-18). There also currently exist very few specificpharmacological antidotes to the drugs frequently associated with lifethreatening overdose cases (Moudgil, B. M., Seventh Year Annual Report.2001: Engineering Research Center for Particle Science and Technology,University of Florida).

An important factor influencing drug distribution in the body is theability of toxins to bind to blood proteins and tissues. Certain tissueshave strong binding affinities for specific toxins, causing localizedconcentration in that tissue. This is true especially of the kidney andliver, because of their metabolic and excretory functions. Some toxinsbind noncovalently to albumin, a blood plasma protein, or otherproteins. While bound to protein, the complex becomes pharmacologicallyinert and is trapped in the bloodstream due to its large size. Onlyunbound drugs are able to cross lipoprotein membranes and exert aneffect. A drug's free molecule concentration is likely to increaseduring an overdose, since protein-binding sites are more readilysaturated. Therefore, it is expected that a patient with low levels ofalbumin will experience higher toxicity effects than a patient withnormal levels (Lu, F., Basic Toxicology: Fundamentals, Target Organs,and Risk Assessment. 3rd ed. 1996, Taylor and Francis: Washington;Fenton, J. J., Toxicology: A Case-Oriented Approach. 2002, CRC Press:Boca Raton; Stine, K. E. and T. M. Brown, Principles of Toxicology.1996, CRC Press: Boca Raton).

Micron-scale and nano-scale core-shell particulate systems, eitherhollow or fluid-filled, have become of recent interest. Core-shellparticles find important applications in encapsulation of a variety ofmaterials for catalysis and controlled release applications (e.g. drugs,enzymes, pesticides, dyes, etc.); for use as filler in lightweightcomposites, pigment, or coating materials; and in biomedical implantmaterials (Putlitz, B. Z. et al., Adv. Mater., 2001, 13:500-+; Walsh, D.and Mann, S., Nature, 1995, 377:320-323; Walsh, D. et al., Adv. Mater.,1999, 11:324-328; Zhong, Z. et al., Adv. Mater., 2002, 12:206-209;Caruso, F., Chem.—Eur. J., 2000, 6:413-419).

Recently, the use of particulate systems as a treatment for patientsoverdosed on lipophilic drugs has been proposed (Moudgil, B. M., SeventhYear Annual Report. 2001: Engineering Research Center for ParticleScience and Technology, University of Florida). Several particulatesystems, including microemulsions, polymer microgels, silica nanotubesand nanosponges, and silica core-shell particles, are currently beinginvestigated for this detoxification purpose. It has been proposed that,when intravenously administered to an overdosed patient, such particleswill effectively detoxify the patient's circulatory system of theparticular lipophilic toxin by either: (a) absorption, from theselective partitioning of the drug molecules from the blood to thehydrophobic core of the particle; or (b) adsorption of the drugmolecules onto surfaces of surface-functionalized particles.Furthermore, in order to catalyze the toxin metabolism, and hence itsremoval from the blood, the immobilization of toxin-specific catabolicenzymes on or within particles is being pursued (Moudgil, B. M., SeventhYear Annual Report. 2001: Engineering Research Center for ParticleScience and Technology, University of Florida).

Fabrication of hollow sphere particles has been accomplished usingvarious methods and materials. In general, three fabrication classes arecurrently employed: sacrificial cores, nozzle reactor systems, andemulsion or phase separation techniques (Caruso, F., Chem.—Eur. J.,2000, 6:413-419; Wilcox, D. L. and Berg, M., in Materials ResearchSociety, 1994, Boston: Materials Research Society). The first involvesthe coating of a core substrate with a material of interest, followed bythe removal of the core by thermal or chemical means. In this manner,hollow particles of yttrium compounds (Kawahashi, N. and Matijevic, E.,J. Colloid Interface Sci., 1991, 143:103-110), TiO₂ and SnO₂ (Zhong, Z.et al., Adv. Mater., 2002, 12:206-209), and silica (Caruso, F.,Chem.—Eur. J., 2000, 6:413-419) have been synthesized. Nozzle reactorsystems make use of spray drying and pyrolysis, and their use hassuccessfully led to the fabrication of hollow glass (Nogami, M. et al.,J. Mater. Sci., 1982, 17:2845-2849), silica (Bruinsma, P. J. et al.,Chem. Mater., 1997, 9:2507-2512), and TiO₂ (Iida, M. et al., Chem.Mater., 1998, 10:3780) particles. Emulsion-mediated procedures, orhollow particle synthesis, is a third common method. This has been usedto form latex (Putlitz, B. Z. et al., Adv. Mater., 2001, 13:500-+),polymeric (Pekarek, K. J. et al., Nature, 1994, 367:258-260), and silicacore-shell particles (Underhill, R. S. et al., Abstracts ofpapers of theAmerican Chemical Society, 2001, 221:545).

Calcium carbonate coated core-shell particles have also beensynthesized. By coating polystyrene beads with calcium carbonate,followed by removal of the polymer core, hollow particles in the 1 μm to5 μm size range have been generated (Walsh, D. and Mann, S., Nature,1995, 377:320-323; U.S. Pat. No. 5,756,210). Core-shell particles havealso been synthesized using water-in-oil (Walsh, D. et al., Adv. Mater.,1999, 11:324-328; Enomae, T., Proceedings of the 5^(th) Asian TextileConference, 1999, 1:464-467), and water-in-oil-in-water (Hirai, T. etal., Langmuir, 1997, 13:6650-6653; Hirai, T. and Komasawa, I., KagakuKogaku Ronbunshu, 2001, 27:303-313) emulsions as templates for calciumcarbonate nucleation. In other processes, Lee et al. (Lee, I. et al.,Adv. Mater., 2001, 13:1617-1620) and Qi et al. (Qi, L. M. et al., Adv.Mater., 2002, 14:300) respectively use monolayer-protected goldparticles and double-hydrophilic block copolymer (DHBC)-surfactantcomplex micelles as templates for calcium carbonate deposition,resulting in core-shell particles up to 5 μm in diameter.

Some of the calcium carbonate core-shell systems discussed in thescientific literature are generated by using a biomimetic process(Walsh, D. and Mann, S., Nature, 1995, 377:320-323; Walsh, D. et al.,Adv. Mater., 1999, 11:324-328; Hirai, T. et al., Langmuir, 1997,13:6650-6653; Hirai, T. and Komasawa, I., Kagaku Kogaku Ronbunshu, 2001,27:303-313; Qi, L. M. et al., Adv. Mater., 2002, 14:300). Mineralizationin biological systems has been the focus of intense research becausetheir successful mimicry has important implications for the syntheticdesign of superior materials. Exquisite control of mineral deposition inbiosystems is thought to occur partly due to the presence of aninsoluble organic matrix, along with modulation of the crystal growthprocess via soluble macromolecular species, such as acidic proteins andpolysaccharides (Lowenstam, H. A. and Weiner, S., On Biomineralization,Oxford University Press: New York, 1989).

As can be understood from the above, there remains a need for aparticulate system that is capable of neutralizing or eliminating toxiclevels of drugs within a patient in a short period of time, and whichcan be produced with the high degree of control associated withbiomimetic processes.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to novel materials and methods for use indelivering and segregating substances, such as pharmacological agents,within a patient. One aspect of the invention is directed towardsparticles having a core encapsulated by a solid calcium carbonate shell,with an intermediate layer of amphiphilic molecules surrounding thecore. When the particles of the subject invention are administered to apatient, they are capable of removing lipophilic drugs by absorption ofthe drug through their porous mineral shell and into their core. In oneembodiment, the core of the particles is hollow. In another embodiment,the core contains a fluid, which is preferably an oil. The particles ofthe subject invention can also be administered to a patient as drugdelivery vehicles. Thus, in another aspect, the subject inventionconcerns a method of delivering or sequestering pharmacological agentsby administering the calcium carbonate-encapsulated particles of thesubject invention to a patient in need of such administration.

The particles of the subject invention can be designed with variousporosities, in order to effectively absorb or release a selectedsubstance over a period of time.

In another aspect, the subject invention concerns a method for makingthe calcium carbonate core-shell particles of the subject invention byusing a polymer-induced liquid-precursor (PILP) process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show reactants and resulting core-shell particles of thesubject invention. FIG. 1A shows the amphiphilic nature of stearic acid.FIG. 1B shows reactants utilized to form core-shell particles of thesubject invention. FIG. 1C shows the structure of a core-shell particleof the subject invention.

FIGS. 2A and 2B show polarized light micrographs of thin calciumcarbonate films deposited under stearic acid monolayers via a PILPprocess. Bar=100 μm.

FIGS. 3A-3C show cross-polarized light micrographs with gypsum waveplate of calcium carbonate-coated emulsion droplets. Bar=200 μm.

FIGS. 4A-4D show scanning electron micrographs (SEMs) (FIG. 4A-4C) ofcalcium carbonate-coated core-shell particles and energy dispersivespectroscopic (EDS) data (FIG. 4D). Particles in FIG. 4B were crushed byshearing between glass slides, and show the shell thickness of thoseparticles that were fractured. Bar=10 μm in FIGS. 4A and 4B; and 2 μm inFIG. 4C.

FIG. 5 shows in vitro uptake of amitriptyline (AMT) by CaCO₃ coatedcore-shell particles from saline solutions.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns particles having a core contained withina solid calcium carbonate shell, with an intermediate layer ofamphiphilic molecules surrounding the core. The subject invention alsoconcerns a method of producing the calcium carbonate-encapsulatedparticles by templating a porous calcium carbonate shell onto thesurface of oil-in-water emulsion droplets using a polymer-induced liquidprecursor (PILP) process. In another aspect, the subject inventionpertains to methods for sequestering lipophilic agents within a patientby administering an effective amount of the core-shell particles to thepatient.

Briefly, the particles of the present invention can be produced byblending an oil, such as n-dodecane, with water and an amphiphile, thelatter acting as a surfactant to stabilize the droplets within water,forming emulsion droplets. The resultant emulsion droplets can then beintroduced into solutions of CaCl₂, MgCl₂, and a short-chained acidicpolymer additive (such as polyaspartic acid). A CO₃ ²⁻ counter ion isthen introduced into the mixture, such as by slow peristaltic pumps,thereby producing calcium carbonate coated particles that can then becentrifuged and dried.

The particles can be constructed in sizes suitable for particularapplications, such as micro-scale or nano-scale particles. For example,the process of the subject invention can produce particles, such asmicrospheres, having a calcium carbonate shell within the range of about1 μm to about 200 μm in diameter. In another embodiment, the shell has adiameter within the range of about 1 μm to about 50 μm in diameter. Inyet another embodiment, the shell has a diameter within the range ofabout 1 μm to about 5 μm in diameter. In order to pass through thecirculatory system of the body, smaller particles can be constructedhaving a diameter within the range of about 100 nm to about 300 nm, forexample, using microemulsion templates.

Advantageously, the method of the subject invention can produceparticles having a shell of uniform thickness. Preferably, the calciumcarbonate shell has a thickness within the range of about 100 nm toabout 1000 nm in thickness. The core-shell particle of claim 1, whereinsaid shell has a thickness within the range of about 200 nm to about 500nm in thickness.

The particles of the subject invention are biodegradable and can beadministered to patients for sequestration of a pharmacological agent(drug uptake) following an overdose, as a detoxification agent.Detoxification can occur through several mechanisms. Once administeredinto a patient (e.g., via the circulatory system), the particles canabsorb a lipophilic drug into their oily core, or adsorb the drugthrough dipole/charge interactions with the mineral shell. Optionally,drug-degrading enzymes, such as P450 enzymes, can operate within theparticles, or be coated onto or otherwise associated with the surface ofthe particles. In another embodiment, the particles can release enzymesthat degrade the drug into harmless catabolites. The calcium carbonateshell provides stabilization to the emulsion, and operates as amolecular screen or filter, to avoid saturation of the particles withproteins and other lipophilic species in the blood, for example. Theparticles of the subject invention can also be administered to a patientas drug delivery vehicles, such as controlled release drug deliveryvehicles, which could occur through either pores templated into theshell, or via degradation of the shell.

Preferably, the particles are of nano-scale dimensions andnon-aggregating, to avoid blockage of blood capillaries (if administeredinto the circulatory system), and are biocompatible (e.g.,non-thrombogenic). If the particles are not sufficiently small to passthrough the blood-renal barrier, a biodegradable material can beincluded for gradual removal of the particulates from the blood stream(at a rate slow enough for the body to tolerate the gradual release ofthe absorbed toxin). Optionally, environment-sensitive catabolic enzymesfor catalysis of the target drug are immobilized within the particles;in which case, the synthesis can be accomplished under benign processingconditions.

The subject invention also concerns a method of producing the calciumcarbonate-encapsulated particles of the subject invention using apolymer-induced liquid-precursor (PILP) process. Using the novel andfacile method of the subject invention, calcium carbonate “hard”shell—“soft” core particles can be synthesized under benign conditions.The method of the subject invention utilizes an oil-in-water emulsiondroplet as a template. The procedure relies on the surface-induceddeposition of a calcium carbonate mineral precursor on to emulsiondroplets by a polymer-induced liquid-precursor (PILP) process, elicitedby including short-chained highly acidic polymers, such as polyasparticacid, into crystallizing solutions of calcium carbonate which are slowlyraised in supersaturation. The deposition of thin films of calciumcarbonate onto glass coverslips using the PILP process has beendemonstrated, as described previously (Gower, L. B. and Odom, D. J., J.Cryst. Growth, 2000, 210:719-734). In those studies, in situobservations revealed that the acidic polymer transforms the solutioncrystallization process into a precursor process by inducingliquid-liquid phase separation in the crystallizing solution. Dropletsof a liquid-phase mineral precursor can be deposited onto varioussubstrates in the form of a film or coating, which upon solidificationand crystallization, produces a continuous mineral film that maintainsthe morphology of the precursor phase (hence, the name precursor). Usingthe method of the subject invention, the PILP process is utilized tocoat an oil droplet in solution, generating a fluid-filled core-shellparticle with a thin uniform shell of calcium carbonate. In some cases,the precursor phase may not appear to be a liquid, but instead havesolid-like characteristics (e.g. glassy). In either case, the importantaspect is that both are an amorphous precursor phase, which due tocoalescence during the formation of the phase, lead to a smoothcontinuous coating of mineral rather than the traditional solutioncrystallization of three-dimensional crystallites. It has also beenfound that the inhibitory action of Mg-ion can lead to a similarprecursor process, and in the presence of surfactant, polymer may not benecessary, although optimal conditions include a combination of Mg-ionand polymer.

The process of the subject invention can be carried out under a varietyof conditions. For example, in the case of an aqueous system, theprocess can be carried out at a temperature of about 4° C. to about 28°C. For ease of processing, the process can be carried out at roomtemperature (about 23° C.). The process is preferably carried out at apH within the range of about 7 to about 11 and at 1 atm. Morepreferably, the process is carried out at a pH of about 11. However, theprocess can be carried out at a pH lower than 7 or higher than 11provided a surfactant is utilized that remains charged at the particularpH. Preferably, the oil:water ratio is within the range of about 1:8 andabout 1:10, by volume. More preferably, the oil:water ratio is about1:9, by volume.

Using the process of the subject invention, the diameter of theparticles can be controlled. For example, the diameter of the particlescan be increased by increasing the size of the emulsion droplet fromwhich the particles are formed. Vesicular types of particles (such asunilamellar or multilamellar liposomes) are feasible as well, whichcould be used to fabricate core-shell particles with an aqueous interiorsurrounded by the mineral shell. For example, in preparing theparticles, a liposome could be substituted for the emulsion droplet as atemplate, which would then be exposed to the amorphous mineralprecursor. This could increase the potential number of applications toinclude encapsulated agents that require an aqueous environment, such aswater soluble molecules and macromolecules, biopolymers (e.g. proteins,DNA) and cells.

Using the process of the subject invention, the calcium carbonate shellporosity can be controlled. Because the highly PILP phase willpreferentially deposit on charged or polar regions of patternedsubstrates, it is possible to pattern porosity into the mineral shell byusing an organic template with hydrophobic domains. For example,increased porosity can be obtained by increasing the quotient ofsurfactant with uncharged head groups (such as cholesterol or diolein)in the mixture of surfactants used to stabilize the emulsion droplet.

One or more of a variety of short-chained acidic polymers can beutilized to initiate the amorphous liquid-phase mineral precursor,including different polymers and biological materials. As used herein,the term “short-chained acidic polymer” is intended to meanoligomeric-length scale polymers bearing at least one acidicfunctionality on one or more monomers of the polymer chain. Polyacrylicacid (PAA), polymethacrylates (PMA), sulfonated polymers, phosphorylatedpeptides and polymers, sulfated glycoproteins, polyaspartic acid,polyglutamic acid, and copolymers of these materials can be utilized toinduce the liquid-phase separation, for example. A range of polymermolecular weights can be suitable if the other variable of thecrystallizing conditions are appropriately modified to generate the PILPphase.

Unlike those particles reported previously, using the method of thesubject invention allows one to generate a smooth and uniform shell ofcalcium carbonate around the oil droplet, and not an aggregation ofindividual crystals, as is common among the previously published work.Furthermore, in this manner, oil can be encapsulated within theparticle, leading to a “soft” fluidic core—a feature that isadvantageous (although not necessary) for the effective extraction oflipophilic molecules from aqueous media by an absorption mechanism.

Preferably, the shell of the particle of the subject invention iscomposed of magnesium-bearing calcium carbonate and is at least 80%calcium carbonate. More preferably, the shell is composed of at least90% calcium carbonate. The Mg-ion is added as an additional inhibitoryagent (to eliminate traditional solution crystallization), andpotentially other ions or molecules could serve this function, incombination with the polymer.

The core of the core-shell particle is a void containing a compound inthe oil phase that is incompatible with water. Preferably, the compoundin the oil phase is a hydrophobic compound, such as an oil. Morepreferably, the hydrophobic compound is an organic compound having asolubility to water of not more than 1 gram per 10 grams of water at 20°C. For example, one or more of a variety of oils, such as dodecane orhexadecane, can be incorporated within each particle, occupying itshollow core. Other organic compounds that can be utilized include, butare not limited to, cyclohexane, n-hexane, benzene, cottonseed oil,rapeseed oil, squalane, squalene, waxes, styrene, divinylbenzene, butylacrylate, 2-ethylhexyl acrylate, cyclohexyl acryalate, decyl acrylate,lauryl acrylate, dodecenyl acrylate, myristyl acrylate, palmitylacrylate, hexadecenyl acrylate, stearyl acrylate, octadecenyl acrylate,behenyl acrylate, butyl methacrylate, 2-ethylhexyl methacrylate,cyclohexyl methacrylate, decyl methacrylate, lauryl methacrylate,dodecenyl methacrylate, myristyl methacrylate, palmityl methacrylate,hexadecenyl methacrylate, stearyl methacrylate, octadecenylmethacrylate, behenyl methacrylate, silicone macromonomers, and thelike.

Particles can be loaded with a selected substance or substances, such asa biologically active agent, by contact with a solution containing theagent. In one embodiment, the biologically active agent, such as adetoxifying enzyme, is incorporated within the emulsion droplet duringformation of the core-shell particle. Loading can be carried out byadding the biologically active agent to the oil phase prior toemulsification and coating of the droplet, for example. Becausedetoxifying enzymes are typically oil soluble, they can be readilycaptured into the oil-in-water emulsion prior to encapsulation with themineral shell.

In another aspect, the subject invention pertains to a method ofsequestering a lipophilic agent within a patient by administering aneffective amount of core-shell particles to the patient, wherein thecore-shell particles absorb the lipophilic agent through their calciumcarbonate shell and into their oil core. The particles can beadministered through any of a variety of routes known in the art,including enteral and parenteral, such as intravenous. Preferably, theparticles are administered into the circulatory system of the patient,via a blood vessel, such as a vein or artery. The patient may besuffering from overdose, wherein a toxic concentration of the lipophilicagent is present within the patient, such as in the bloodstream. Thepatient may also be suffering from harmful drug interaction between thelipophilic agent and another lipophilic agent or non-lipophilic agent.

In another aspect, the subject invention pertains to a method ofdelivering a biologically active agent to a patient by administering aneffective amount of core-shell particles containing a selectedbiologically active agent to the patient, wherein the core-shellparticles can release the biologically active agent within the patient.The particles can be administered through any of a variety of routesknown in the art, including enteral, pulmonary, and parenteral, such asintravenous. Preferably, the particles are administered into thecirculatory system of the patient, such as through a blood vessel.

The particles of the subject invention can be administered using any ofa variety of means known in the art. For example, administration of aneffective amount of particles can include the injection of the particlesin a blood vessel, such as an artery.

Following administration of the particles and drug release or drugsequestration, the spent particles can, optionally, be retrieved fromthe patient using a variety of methods. For example, if the particlesare not sufficiently biodegradable, they can be filtered from the blood,such as in a dialysis process.

The term “biodegradable”, as used herein, means capable of beingbiologically decomposed. A biodegradable material differs from anon-biodegradable material in that a biodegradable material can bebiologically decomposed into units which may be either removed from thebiological system and/or chemically incorporated into the biologicalsystem.

The term “biocompatible”, as used herein, means that the material doesnot elicit a substantial detrimental response in the patient. It shouldbe appreciated that when a foreign object is introduced into a livingbody, that the object may induce an immune reaction, such as aninflammatory response that can have negative effects on the patient. Asused herein, the term “biocompatible” is intended to include thosematerials that cause some inflammation, provided that these effects donot rise to the level of pathogenesis.

The particles of the subject invention can be used as a vehicle for thedelivery of biologically active agents, such as medical substances inthe field of therapeutics. The active agents may be incorporated in theoil-containing core or chemically bonded to the calcium carbonate shell,for example.

As used herein, the terms “incorporated within” or “otherwise associatedwith” mean that the particular agent is contained within the particle ofthe subject invention or is directly or indirectly bound to the particlein some fashion. For example, the biologically active agent can becontained within the oil core of the particle, or operate as a componentof the calcium carbonate shell or amphiphilic layer. The biologicallyactive agent can be “free” or bonded to any of the other components ofthe particle. The particular agent can be incorporated within, orotherwise associated with, the particles of the subject invention,during or subsequent to production of the particles. For example, abiologically active agent, such as an enzyme, can be attached to theouter shell through direct adsorption or through a linker molecule.Alternatively, the agent can be physically entrapped in the mineralphase, as it is deposited, and subsequently released upon degradation ofthe mineral.

The biologically active agents that can be delivered using the particlesof the subject invention can include, without limitation, medicaments,vitamins, mineral supplements, substances used for the treatment,prevention, diagnosis, cure or mitigation of disease or illness,substances which affect the structure or function of the body, or drugs.The active agents include, but are not limited to, antifungal agents,antibacterial agents, anti-viral agents, anti-parasitic agents, growthfactors, angiogenic factors, anaesthetics, mucopolysaccharides, metals,cells, antibodies, antibody fragments, and other agents. Because theprocessing conditions can be relatively benign, live cells can beincorporated into the particles during their formation, or subsequentlyallowed to infiltrate the particles through tissue engineeringtechniques.

The terms “pharmaceutically active agent”, “biologically activecompound”, “biologically active agent”, “active agent”, “activecompound” and “drug” are used herein interchangeably and includepharmacologically active substances that produce a local or systemiceffect in a human or non-human animal. The terms thus mean any substanceintended for use in the diagnosis, cure, mitigation, treatment orprevention of disease or in the enhancement of desirable physical ormental development and conditions in a human or non-human animal.

Examples of antimicrobial agents that can be delivered using theparticles of the present invention include, but are not limited to,isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine,rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin,azithromycin, clarithromycin, dapsone, tetracycline, erythromycin,cikprofloxacin, doxycycline, ampicillin, amphotericine B, ketoconazole,fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin,pentamidine, atovaquone, paromomycin, diclarazaril, acyclovir,trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir,iatroconazole, miconazole, Zn-pyrithione, and silver salts, such aschloride, bromide, iodide, and periodate.

Growth factors that can be incorporated into or otherwise associatedwith the particles of the present invention include, but are not limitedto, basic fibroblast growth factor (bFGF), acidic fibroblast growthfactor (aFGF), nerve growth factor (NGF), epidermal growth factor (EGF),insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), platelet-derivedgrowth factor (PDGF), tumor angiogenesis factor (TAF), vascularendothelial growth factor (VEGF), corticotropin releasing factor (CRF),transforming growth factors alpha and beta (TGF-α and TGF-β),interleukin-8 (IL-8), granulocyte-macrophage colony stimulating factor(GM-CSF), bone morphogenic protein (BMP), the interleukins, and theinterferons.

Other agents that can be incorporated into or otherwise associated withthe particles of the subject invention include acid mucopolysaccharidesincluding, but not limited to, heparin, heparin sulfate, heparinoids,dermatan sulfate, pentosan polysulfate, chondroitin sulfate, hyaluronicacid, cellulose, agarose, chitin, dextran, carrageenin, linoleic acid,and allantoin.

Proteins that can be incorporated into or otherwise associated with theparticles of the subject invention include, but are not limited to,collagen (including cross-linked collagen), fibronectin, laminin,elastin (including cross-linked elastin), osteonectin, bonesialoproteins (Bsp), alpha-2HS-glycoproteins, bone Gla-protein (Bgp),matrix Gla-protein, bone phosphoglycoprotein, bone phosphoprotein, boneproteoglycan, protolipids, bone morphogenetic protein, cartilageinduction factor, platelet derived growth factor and skeletal growthfactor, or combinations and fragments thereof.

Other biologically active agents that can be incorporated into orotherwise associated with the particles of the subject invention includegenetically-modified or non-genetically modified cells. Thus, theparticles of the subject invention can contain such cells within theircore and be administered to a patient. The cells can be non-stem cells(mature and/or specialized cells, or their precursors or progenitors) orstem cells. Thus, the cells can range in plasticity from totipotent orpluripotent stem cells (e.g., adult or embryonic), precursor orprogenitor cells, to highly specialized or mature cells, such as thoseof the pancreas. In one embodiment, the cells are genetically modifiedto produce a biologically active agent, such as a detoxifying enzyme.

Stem cells can be obtained from a variety of sources, including fetaltissue, adult tissue, cord cell blood, peripheral blood, bone marrow,and brain, for example. Stem cells and non-stem cells (e.g., specializedor mature cells, and precursor or progenitor cells) can bedifferentiated and/or genetically modified. Methods and markers commonlyused to identify stem cells and to characterize differentiated celltypes are described in the scientific literature (e.g., Stem Cells:Scientific Progress and Future Research Directions, Appendix E1-E5,report prepared by the National Institutes of Health, June, 2001). Thelist of adult tissues reported to contain stem cells is growing andincludes bone marrow, peripheral blood, brain, spinal cord, dental pulp,blood vessels, skeletal muscle, epithelia of the skin and digestivesystem, cornea, retina, liver, and pancreas.

The active agents incorporated within, or otherwise associated with, theparticles of the subject invention can exhibit modified releasecharacteristics. Release of the active agent can be controlled using avariety of methods. For example, biologically decomposable conjugatescan be utilized. Alternatively, release of the active agent can becontrolled by inserting the active agent in various components of theparticle that have a different biodegradability. For example, if used inmedical or agricultural applications, it may be desired to be able tocontrol the release dosage and release rate of active agents. In oneembodiment, the particles exhibit a decreasing (decaying) rate ofrelease (first-order release kinetics). In another embodiment, theparticles exhibit a constant rate of release (zero-order releasekinetics). In another embodiment, the particles exhibit one or moresudden releases, or bursts, after a certain delay time.

The particles of the subject invention can be utilized to administerhormones, for example. An important field of application is thedevelopment of therapeutic systems for the controlled release of ananti-diabetic agent, such as insulin, in the treatment of pancreaticdiabetes. The particles of the subject invention can also be utilized toadminister anti-tumor compounds, such as cytotoxic agents, for thetreatment of cancer.

Larger micro-scale particles of the invention can contain cells.According to the methods of the invention, such particles can beutilized to deliver the cells, and/or active agents produced by thecells, in vivo. Examples of cells that can be incorporated within, orotherwise associated with, the particles of the subject inventioninclude, but are not limited to, stem cells, precursor or progenitorcells, chondrocytes, pancreatic cells, hepatocytes, and neural cells.Such cells can be released from the particles upon degradation of theshell in vivo.

The surface of the particles can be modified using surface modificationmethods known to those of ordinary skill in the art. For example, theamphiphilic layer composition can be varied to vary the uncharged headgroup domain size.

As used herein, the term “lipophilic” is intended to mean oil soluble.Examples of lipophilic drugs include amitriptyline, bupivicaine, andamiodarone.

As used herein, the term “oil” is intended to mean any nonpolar,water-insoluble compound.

As used herein, the terms “amphiphile” “amphiphilic compound”, and“surfactant” are used herein interchangeably and intended to mean acompound having at least one hydrophilic (polar) portion and at leastone hydrophobic (nonpolar) portion, such as stearic acid and arachidicacid. Typically, amphiphiles exhibit amphiphilic behavior in which theirmolecules become concentrated at the interface between a polar solventand a nonpolar solvent. Preferably, the amphiphilic compounds used inthe subject invention have molecules with at least one hydrophilic headgroup and at least one hydrophobic tail. More preferably, theamphiphilic compound has a partially deprotonated carboxylic acidheadgroup functionality.

The particles of the subject invention can be formulated in any of avariety of forms or shapes in the micro- or nano-scale size range (e.g.,microparticles or nanoparticles). The particles of the present inventioncan be, for example, capsules (e.g., microcapsules or nanocapsules), orspheres (e.g., microspheres or nanospheres).

The core-shell particles of the subject invention can be formulated andadministered as a pharmaceutical composition, containing apharmaceutically acceptable carrier or diluent. The pharmaceuticalcompositions of the subject invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in a number of sources which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Science (Martin E W [1995] Easton Pa., MackPublishing Company, 19^(th) ed.) describes formulations which can beused in connection with the subject invention. Formulations suitable forparenteral administration include, for example, aqueous sterileinjection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules, vials, and disposable syringes made ofglass or plastic, and may be stored in a freeze dried (lyophilized)condition requiring only the condition of the sterile liquid carrier,for example, water for injections, prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterile powder,granules, tablets, etc. It should be understood that, in addition to theingredients particularly mentioned above, the formulations of thesubject invention can include other agents conventional in the arthaving regard to the type of formulation in question. The pharmaceuticalcompositions can be included in a container, pack, or dispenser,together with instructions for administration.

The particles of the subject invention can be applied as a film orcoating on a substrate. The substrate can be composed of any material,such as metal, polymer, and/or ceramic materials.

The term “patient”, as used herein, refers to any vertebrate species.Preferably, the patient is of a mammalian species. Mammalian specieswhich benefit from the disclosed methods of drug delivery and/ordetoxification include, and are not limited to, apes, chimpanzees,orangutans, humans, monkeys; domesticated animals (e.g., pets) such asdogs, cats, guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits,and ferrets; domesticated farm animals such as cows, buffalo, bison,horses, donkey, swine, sheep, and goats; exotic animals typically foundin zoos, such as bear, lions, tigers, panthers, elephants, hippopotamus,rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests,prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena,seals, sea lions, elephant seals, otters, porpoises, dolphins, andwhales.

The particles of the subject invention can be used in novel therapeuticsystems in which ferrous components are associated with the particles soas to impart magnetic properties to the particles. The magneticproperties of the particles can induce and control the release of theactive agent via a “magnetic switch” that may be operated from outsidethe body. In some therapeutic approaches, systems of particles andactive agents can be selectively accumulated in their target area usingexternal magnetic fields. For treating very special problems, smallmagnets can be implanted within the patient for local control in thetarget area, e.g., a tumor area.

The particles of the subject invention are useful in diagnosticapplications, as well. For example, the particles of the subjectinvention can incorporate, or otherwise be associated with,visualization markers, and are applicable for many special indicationssuch as magnetic resonance (MR) lymphography after intravenous or localinterstitial administration, tumor visualization, visualization offunctions or malfunctions, of plaque (atherosclerosis imaging), thrombiand vascular occlusions, MR angiography, perfusion imaging, infarctvisualization, visualization of endothelial damages, receptor imaging,visualization of blood-brain barrier integrity, etc., as well as fordifferential diagnosis, in particular, for distinguishingtumors/metastases from hyperplastic tissue.

The particles are also useful for industrial applications, such as useas light-weight pigment/filler particles or as platelets for highcontrast print gloss.

The terms “comprising”, “consisting of”, and “consisting essentially of”are defined according to their standard meaning and may be substitutedfor one another throughout the instant application in order to attachthe specific meaning associated with each term.

MATERIAL AND METHODS

Emulsion Substrate Svnthesis. Oil-in-water emulsion droplets weresynthesized by blending in a household kitchen blender, n-dodecane oil(SIGMA-ALDRICH) and distilled water in 1:9 volume ratio, stabilized with1% w/v stearic acid (SIGMA-ALDRICH) (per oil phase volume). Thedistilled water was adjusted to the desired pH using 0.1M NaOH (FISHERSCIENTIFIC) prior to emulsification.

Particle Synthesis. Immediately after preparing the emulsion, asindicated above, 1 mL of the emulsion was pippetted into 35 mm FALCONpolystyrene petri dishes, followed by 1 mL of an 80 mM/400 mMCaCl₂/MgCl₂ solution (SIGMA-ALDRICH) (freshly prepared using distilledwater, and filtered by 0.2 μm ACRODISC syringe filters). Next, 36 μL ofa freshly prepared and filtered 1 mg/mL solution ofpoly-(α,β)-D,L-aspartic acid (MW 8600) (ICN/SIGMA-ALDRICH) wastransferred to each petri dish by micropipet. The petri dishes were thencovered by parafilm, which was punched with a small hole, into which theoutflow end of the tubing from an ultra-low flow peristaltic pump(FISHER SCIENTIFIC) was inserted. At a rate of approximately 0.025mL/min, 2 mL of a freshly prepared and filtered solution of 300 mM(NH₄)₂CO₃ (SIGMA-ALDRICH) was pumped into each petri dish (taking about80 minutes to complete). The resulting product was collected andcentrifuged at 8000 rpm for 10 minutes, rinsed with saturated CaCO₃(SIGMA-ALDRICH), then re-centrifuged under the same conditions. After arinsing with ultrapure ethanol (FISHER SCIENTIFIC), the product wasre-centrifuged a final time under the same conditions, and then left todry in air overnight.

Determination of Particle Morphology and Composition. The driedparticles were examined by an OLYMPUS BX60 polarized light microscope,using a gypsum wave-plate in order to observe both amorphous andcrystalline phases. For scanning electron microscopy (SEM) observations,particle samples were spread onto aluminum studs, and then gold-coatedand examined with a JEOL 6400 SEM. Energy Dispersive Spectroscopy (EDS)was used for elemental composition analysis of the particle shell. Fordiffraction studies, dried particles were adhered to double-sided tape,and analyzed in a PHILIPS APD 3720 X-ray instrument.

EXAMPLE 1 Formation of Free-Standing Films of Calcium Carbonate UnderLangmuir Monolayers

As a preliminary step to core-shell particle fabrication, and to betterunderstand the deposition of calcium carbonate films on surfactanttemplates, the formation of freestanding films of the mineral underLangmuir monolayers spread at the air-liquid interface was investigated.FIGS. 2A and 2B show polarized light micrographs of mineral filmsdeposited under stearic acid monolayers. The micrographs were takenusing a gypsum wave plate, which renders amorphous material to appear asthe same magenta color as the background. As seen by the lack ofbirefringence in FIG. 2A, the initial film is amorphous and opticallyisotropic (iso). Interestingly, the film cracked like a brittle glasswhen scooped onto a coverslip, which is not typical for an amorphouscalcium carbonate (ACC) phase (granular ACC precipitates are producedfrom highly supersaturated solutions). If the films are removed fromsolution and let to dry in air, they crystallize in either spherulitic(sph) or single-crystalline (sc) patches (FIG. 2B). Similar results wereobtained under arachidic acid monolayers.

Repeating this experiment using cholesterol or diolein surfactants, incontrast, did not yield the uniform mineral film under the monolayer.Both stearic acid and arachidic acid surfactants have partiallydeprotonated carboxylic acid headgroup functionalities, whilecholesterol and diolein surfactants, which bear alcohol moieties, remainpolar but uncharged. Therefore, the surface charge on the monolayer isthought to play an important role in attracting mineral species and theion-binding polymer to the surface, serving to increase ion saturation,and induce the deposition of the mineral precursor.

EXAMPLE 2 Surface-Induced Deposition of a Mineral Shell onto a ChargedEmulsion Droplet

Using stearic acid as a surfactant, n-dodecane oil was dispersed inwater to form an oil-in-water emulsion. To coat these emulsion droplets,they were first combined with Ca²⁺ dissolved in aqueous solution, alongwith polyaspartic acid to induce the PILP process. Mg²⁺ ions were alsoadded to enhance the inhibitory action of the polymer, which helps toinhibit traditional crystal growth from solution (as opposed to from theprecursor phase). The CO₃ ²⁻ counterion was subsequently pumped into theabove mixture using ultra-low-flow peristaltic pumps. To monitor itseffect on mineral deposition, the surface charge on the surfactant layerwas varied by adjusting the pH of the aqueous solutions between 7 and 11(pK_(a) of stearic acid is 10.15).

FIG. 3A shows freshly coated particles synthesized in this manner at pH7. As detected from the lack of birefringence under cross-polarizedlight, the particles, as expected, initially had an amorphous CaCO₃shell. After rinsing the particles with saturated CaCO₃ and ethanol, theparticles were allowed to dry in air. FIG. 3B shows particlessynthesized at pH 8 that were allowed to age in air for 1 week. Thepresence of birefringence in some of the spherical shells can now bedetected, indicating an amorphous to crystalline phase transformationhad taken place in the mineral shell, as was observed in the thin flatfilms. Furthermore, the Maltese cross pattern in the birefringence (seeFIG. 3C, which is a magnification of FIG. 3B, lower right) indicates aspherulitic crystalline structure of the shell. The polycrystallinenature of spherulites suggests that the shell is naturally porous(without requiring patterning of the deposition process), although it isat a very fine scale since the particles appear smooth at relativelyhigh magnification.

Under scanning electron microscopy (SEM), the morphology and uniformityof the particles were better judged. From these observations, particlessynthesized at pH 11 yielded the best results—fairly monodisperse,uniformly spherical particles of diameter ranging between 1-5 μm (seeFIG. 4A). Since the formation of a shell around the emulsion dropletswas most enhanced at this pH setting, the increased surface charge at pH11, compared to lower pHs, was deemed to be instrumental in thedeposition of the PILP precursor. A sample of these particles wassheared between glass slides, and SEM of the resulting product ispictured in FIG. 4B. The presence of spherical shell fragments andhollow cores confirms the core-shell structure of the fabricatedparticles. The shell thickness, based on SEM images such as those shownin FIGS. 4A-4C, was observed to be between 200 and 500 nm in thickness.No specific correlation between particle diameter and shell thicknesswas noticed, although it is thought that by controlling the reactiontime, this property might be tailorable. The shell is a smooth uniformcoating (FIG. 4C), and although it appears to be spherulitic, it is notcomposed of an aggregation of individual crystals that nucleated fromsolution on the template, but rather it transformed from an amorphousprecursor phase. Using Energy Dispersive Spectroscopy (FIG. 4D), thepresence of Ca, Mg, and O in the particle shell was confirmed,suggesting that the mineral is a Mg-bearing CaCO₃ phase (which is alsoseen for PILP films deposited onto solid substrates).

In summary, the synthesis of core-shell particles was carried out usingan oil-in-water emulsion as a substrate. The reaction chemistry wasconducted at a consistent final Ca²⁺ and CO₃ ²⁻ concentration of 20 mMand 150 mM respectively, with a polymer level varied between 0 and 300μg/ml, and a Mg level varied between 0 and 100 mM. Peristaltic pumpingwas employed to introduce the CO₃ ²⁻ counterion into the reactioncontainer. This pumping technique was utilized to synthesize core-shellparticles under the following conditions: Ca/Mg=20/100 mM; CO₃ ²⁻=150mM; polymer=10 μg/ml; and a pH 11 environment. Since these conditionsseemed to yield the best particles, further testing was conducted onparticles fabricated under these conditions.

EXAMPLE 3 Deposition of Mineral Shell in Absence of PILP-EnhancingPolymer

When a PILP-enhancing polymer was not included in the reacting solution,core-shell particles could, under certain conditions, still besynthesized successfully. In some tests, the particles made withoutpolymer were indistinguishable from those made with polymer underoptical microscopy. Under SEM, however, the particles formed withoutpolymer did not always match the quality of those made with polymer—aportion of the product was not uniformly spherical but of some modifiedamorphous shape. The reason these particles formed even in the absenceof polymer is most likely due to the relatively high amount of Mg used(at a Ca to Mg ratio of 1 to 5). Mg is a potent crystal growth inhibitorand may have elicited a PILP-like mechanism in the formation of theshell.

A series of tests were therefore conducted, varying both the polymer andMg levels in the synthesis procedure, to determine their effects. In theabsence of Mg, no particles formed at all. Instead, polymer-modifiedcrystals were abundant at all levels of polymer (tested between 10 and300 μg/ml). In a second set of experiments, polymer concentration wasmaintained at either 10 μg/ml or 100 μg/ml, and the final concentrationof Mg in the reacting solution was varied between 20 and 100 mM. In thiscase, core-shell particles were successfully synthesized at Mg levels aslow as 20 mM, and at polymer concentrations of 10 μg/ml.

Apparently, a small amount of Mg (at 1:1 ratio of Ca/Mg) is necessary topromote the formation of a core-shell particle. However, increasedpolymer level significantly perturbed the process. Particles formed atMg=80 mM and at a polymer concentration of 10 and 100 μg/ml,respectively, were compared. At 100 μg/ml, the particles formedpoorly—without uniformity in shape or sizes, while the lower polymersamples formed normally. This trend held true at every Mg level tested(20, 40, 60, and 80 mM)—the particles with higher polymer doses did notform as well as with lower polymer doses. Since more polymer is likelyto better inhibit mineral nucleation, the higher doses may not haveallowed a shell to deposit or solidify very well on the emulsiondroplet. In addition, because of the charge associated with the acidicpolymer (especially at the pH 11 condition of the experiment), the highpolymer level may have compromised the stability or function of theemulsion droplet.

EXAMPLE 4 Degradability of Particles Svnthesized using PILP andPILP-Enhancing Polymer

Because the shells of the core-shell particles are generated via PILP,they are in a metastable amorphous state. This suggests that theparticle shells are susceptible to biodegradation once reintroduced intothe blood. This is considered an important advantage of a CaCO₃core-shell particle for use in drug detoxification, as it facilitatesthe removal of particle components from the blood stream. To determinewhether these particles are indeed degradable, samples of driedparticles were dispersed in buffered saline solutions, and monitored forseveral weeks. The particles were continually stirred while in solutionto simulate the constant agitation expected if they were flowing withinthe circulatory system.

Particles dispersed in phosphate buffered saline solutions (PBS) (pH˜7.4) at a concentration of approximately 4 mg/mL of and 16 mg/mL losttheir spherical shape due to dissolution as early as a week afterdispersion, and the remaining material eventually recrystallized intoseveral crystal morphologies. “Concentration” in this case is defined asmg of particles per mL of solution (saline, blood, etc.), and the testedconcentrations are within the range that is proposed for detoxificationof an overdosed patient. While the particle shell is degradable, thecomponent materials remained as insoluble precipitates if notsufficiently diluted. In the blood stream, the reprecipitation is lesslikely since the larger volume will dilute the ionic species createdduring particle degradation.

EXAMPLE 5 Drug-Uptake Efficiency of Particles Synthesized using PILP andPILP-Enhancing Polymer

To determine whether these particles were capable of uptaking lipophilicdrugs, High Performance Liquid Chromatography (HPLC) was employed. Thetest drug used for these studies was amitriptyline (AMT). AMT is themost widely prescribed tricyclic anti-depressant (TCA) in the UnitedStates. The drug is a significant cause for hospitalizations due totoxicity and has been reported as the most common cause of drug relateddeaths and suicide. Other drugs in this class include clomipramine,desipramine, imipramine, norclomipramine, nortriptyline, andtrimipramine, but AMT is more typically prescribed. AMT is a highlylipophilic drug and is thought to effectively treat depression byblocking the physiological inactivation of biogenic amines.

Particles were introduced into saline solutions isotonic to blood atconcentrations of 0.01%, 0.025%, and 0.05% (1%=1 mg particles/10 mLsolution). AMT was then added and concentrated to 1 mM in the mixture.That mixture was sonicated for 5 minutes and then filtered bycentrifugation for 30 minutes, during which time the particles werepresumably absorbing the AMT. The amount of AMT remaining in theresulting filtrate was then assessed by HPLC.

Three different samples were tested for comparison purposes. The firstsample was dried core-shell particles containing the oily core. Thesecond sample was core-shell particles that were calcined at 240° C. for1.5 hours while simultaneously vacuum dried to evaporate any n-dodecaneoil in or on the particle (b.p. of n-dodecane=216° C.). From opticalmicroscopy observations, the structure of these treated core-shellparticles remained unaffected. The third sample was commercial CaCO₃obtained from SIGMA-ALDRICH (mostly calcite crystals). The hypothesiswas that particles with oil would absorb significantly larger amounts ofAMT from solution than both particles without oil and the commercialcalcite samples, since the advantage of partitioning the lipophilicmolecules into the oily core was not available to the latter two.

Results of this uptake study are shown in FIG. 5. The particles with oilwere shown to extract 83% of AMT at the lower particle concentrationsand upwards of 97% at 0.025% and 0.05% w/v levels. Surprisingly, theparticles without oil were able to extract just as much drug as theoil-filled particles at the 0.025% and 0.05% levels, and only 10% lessat 0.01%. While the commercial CaCO₃ did absorb the least amount of AMT,the quantity of drug that was extracted from solution was alsounexpectedly high (as high as 50%). The commercial crystals used in thislast sample measured as large as 40μm, and therefore the sample'ssurface area did not compare well to particle samples tested. Therefore,a fourth sample was tested for drug uptake—that of commercial CaCO₃crystals that were mill ground for 18 hours to reduce the crystal size,and therefore increase the surface area, to a range comparable to theparticles samples. In this case, the sample was able to uptake up to 85%of the AMT drug, as shown in FIG. 5. In light of this, it is feasiblethat nano-scale CaCO₃ crystals could uptake over 90% of AMT from salinesolutions. This is an important finding as it suggests that thepartitioning the drug into the oily core of the particle may not be themost prevalent mechanism of drug uptake as initially thought. The drugmay also be adsorbing significantly to the particle or crystal surfaces.

EXAMPLE 6 Drug Detoxification using Calcium Carbonate Core-ShellParticles

The core-shell particles of the subject invention are particularlyuseful for the detoxification of lipophilic drugs within a patient inneed of such detoxification. In one embodiment, an effective amount ofcore-shell particles are administered to the patient, such as throughintravenous injection, wherein the unbound lipophilic drug (e.g.,unbound to blood protein) is simply absorbed through the calciumcarbonate shell of the particles, and into their core, effectivelypartitioning the lipophilic drug from the patient's bloodstream, forexample. The particles can then be allowed to degrade, releasing thelipophilic drug over a period of time that is not harmful to thepatient. Alternatively, the particles can be retrieved from the patientusing known methods of particulate retrieval. In another embodiment, oneor more drug-detoxifying enzymes (also referred to herein as adrug-detoxifying system) are incorporated within, or otherwiseassociated with, the particles of the subject invention. In anotherembodiment, compounds which act as inducers of endogenous drugdetoxifying enzymes can be incorporated within, or otherwise associatedwith, the particles of the subject invention.

Drug biotransformation usually involves two phases, phase I and phaseII. Phase I reactions are classified typically as oxidations,reductions, or hydrolysis of the parent drug. Following phase Ireactions, the metabolites are typically more polar (hydrophilic), whichincreases the likelihood of their excretion by the kidney. Phase Imetabolic products may be further metabolized. Phase II reactions oftenuse phase I metabolites that can catalyze the addition of other groups,e.g., acetate, glucuronate, sulfate, or glycine to the polar groupspresent on the intermediate. Following phase II reactions, the resultantmetabolite is typically more readily excreted. The drug detoxifyingenzymes utilized in the subject invention can catalyze phase Ireactions, phase II reactions, or both phase I and phase II reactions,for example.

Most phase I reactions are catalyzed by the cytochrome P450 (CYP) enzymesystem, which is a superfamily consisting of heme-containing isozymes(van der Weide and, J. and Steijns, L., “Cytochrome P450 Enzyme System:Genetic Polymorphisms and Impact on Clinical Pharmacology”, Ann. Clin.Biochem., 36:722-729, 1999). At least 74 CYP gene families, of which 14are ubiquitous in all mammals, have been described thus far (Nelson, D.R. et al., “P450 Superfamily: Update on New Sequences, Gene Mapping,Accession Numbers, and Nomenclature”, Pharmacogenetics, 6:1-42, 1996).The enzymes belonging to the families CYP1, CYP2, and CYP3 catalyze theoxidative biotransformation of exogenous compounds, including manydrugs, (pro)carcinogens, (pro)-mutagens, and alcohols. Other CYPfamilies are involved in the metabolism of endogenous substances, suchas fatty acids, prostaglandins, and steroid and thyroid hormones.Specific catalytic activities that have been observed with regard tosome P450 isoforms in in vitro assays include testosterone 6-hydroxylaseactivity of CYP3A4, dextromethorphan O-deethyolase activity of CYP2D6,tolbutamide 4-hydroxylase activity of CYP2C9, phenacitin O-deethylaseactivity of CYP1A2, (S)-Mephenytoin 4′-hydroxylase activity of CYP2C19,chorozoxazone 6-hydroxylase activity of CYP2E1, coumarin 7-hydroxylaseactivity of CYP2A6, lauric acid 12-hydroxylase activity of CYP4A11, andpaclitaxel 6-hydroxylase activity of CYP2C8. As indicated above, one ormore P450 enzymes can be incorporated within, or otherwise associatedwith the particles of the subject invention. Alternatively, inducers ofendogenous P450 enzyme activity can be incorporated within, or otherwiseassociated with, the particles of the present invention. For example,there are over twenty different CYP enzymes within the human body, withat least six of the enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1,and CYP3A) accounting for the metabolism of nearly all clinically usefulmedications. Examples of P450 enzymes and their corresponding substratespecificities are listed in Table 1. TABLE 1 Examples of Cytochrome P450(CYP) Enzymes and Corresponding Substrates Enzyme Substrates CYP1A2caffeine, clozapine, fluvoxamine, haloperidol, paracetamol,theophylline, acetominophen, amitriptyline, antipyrine, clomipramine,enoxacin, imipramine, olanzapine, ondansetron, phenacetin, propranolol,tacrine, R(-)warfarin, verapamil CYP2A6 coumarin CYP286 cyclophosphamideCYP2C8 arachidinic acid, paclitaxel, retinoic acid, warfarin CYP2C9cyclophoshamide, diclofenac, hexobarbital, ibuprofen, mefanamic acid,naproxen, phenytoin, piroxicam, tenoxicam, thiotepa, tolbutamide, TCAs,torsemide, S(-)warfarin CYP2C19 amitriptyline, clomipramine, diazepam,heoxbarbital, imipramine, lansoprozole, mephenytoin, mephobarbital,mclobemide, omeprazole, proguarnil, -propranolol CYP2D6 antiarrhythmicssuch as encainide, flecainide, mexiletine, propafenone; antipsychoticssuch as clozapine haloperidol, perphenazine, reduced haloperidol,risperidone, thioridazine; beta-blockers such as bufuralol, metoprolol,propranolol, timolol; opiates such as codeine, dextromethorphan,hydromorphone, methadone, oxycodone, tramadol; TCAs such asamitriptyline, clomipramine, desipramine, imipramine, norclomipramine,nortriptyline, trimipramine; SSRIs such as fluoxetine, paroxetine;miscellaneous antidepressants such as maprotiline, nefazodone,venlafaxine; antihypertensives; debrisoquin;methlenedioxymethamphetamine (Ecstasy); ondansetron; phenformin;sparteine; tacrine; terfenadine; tropisetron; verapamil CYP2E1acetominophen, chlorzoxazone, ethanol, enflurane, halothane, acetone,paracetamol CYP3A4 antiarrhythmics such as amiodarone, lidocaine,propafenone, quinidine; antidepressants such as bupropion, sertraline,TCAs (amitriptyline, clomipramine, desipramine, imipramine,norclomipramine, nortriptyline, trimipramine), venlafaxine;benzodiazepines such as alprazolam, diazepam, midazolam, triazolam;calcium channel blockers such as diltiazem, felodipine, nifedipine,nimodipine, nisoldipine, verapamil; nonsedating antihistamines such asastemizole, terfenadine; acetaminophen; alfentanil; amiodarone; codeine;cyclosporin A/G; carbamazepine; cyclophosphomide; cortisol; dapsone;dexamethasone; dextromethorphan; doxorubicin; erythromycin (N—CH3);ethinylestradial etoposide; fentanyl; felodipine; ifosfamide;lansoprozole; lidocaine; lomustine; lavastatin; omeprazole; ondansetron;progesterone; tamoxifen; taxol; testosterone; triacetyloleandomycin(TAO); vincristine; vinblastine; vinorebine; warfarin; methadoneNote:SSRIs = selective serotonin reuptake inhibitors; TCAs = tricyclicantidepressants

The drug-detoxifying enzyme can be contained within the core of thecore-shell particles or otherwise associated with the particles. Forexample, the drug-detoxifying enzyme can be adsorbed onto the calciumcarbonate shell of the particles.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety, including all figures and tables, to the extent theyare not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

1. A core-shell particle comprising: (a) a shell, wherein said shellcomprises calcium carbonate; (b) a core; and (c) an intermediate layerbetween said shell and said core, wherein said intermediate layercomprises an amphiphilic compound, and wherein said core and saidintermediate layer are surrounded by said shell.
 2. A method for makinga core-shell particle, wherein the method comprises the steps of: (a)preparing a core; and (b) encapsulating the core with a calciumcarbonate shell.
 3. The method according to claim 2, wherein said step(a) comprises forming an emulsion droplet, and wherein said step (b)comprises contacting the emulsion droplet with a calcium-containingsolution.
 4. The method according to claim 3, wherein the emulsiondroplet comprises an oil phase and an amphiphilic compound, and whereinthe core-shell particle comprises: (a) the calcium carbonate shell; (b)the core; and (c) an intermediate layer between the calcium carbonateshell and the core, wherein the intermediate layer comprises theamphiphilic compound, and wherein the core and the intermediate layerare surrounded by the shell.
 5. The method according to claim 4, whereinthe amphiphilic compound has a partially deprotonated carboxylic acidheadgroup functionality.
 6. The method according to claim 4, wherein theamphiphilic compound is selected from the group consisting of stearicacid and arachidic acid.
 7. The method according to claim 3, whereinsaid method further comprises adding a source of Mg ion to thecalcium-containing solution.
 8. The method according to claim 3, whereinsaid method further comprises adding a short-chained acidic polymer tothe calcium-containing solution.
 9. The method according to claim 8,wherein the short-chained acidic polymer is selected from the groupconsisting of polyacrylic acid, polymethacrylate, sulfonated polymer,phosphorylated peptide, phosphorylated polymer, sulfated glycoprotein,polyaspartic acid, polyglutamic acid, and copolymers thereof.
 10. Themethod according to claim 9, wherein the short-chained acid polymercomprises poly-(α,β)-D,L-aspartic acid.
 11. The method according toclaim 8, wherein the short-chained acid polymer is added at aconcentration within the range of about 1 μg/ml and about 100 μg/ml. 12.The method according. to claim 3, wherein said forming an emulsiondroplet comprises contacting a hydrophobic compound with an aqueoussolution.
 13. The method according to claim 12, wherein said forming anemulsion droplet further comprises adding an amphiphilic compound to theaqueous solution.
 14. The method according to claim 13, wherein theamphiphilic compound is selected from the group consisting of stearicacid and arachidic acid.
 15. The method according to claim 3, whereinthe calcium-containing solution comprises CaCl₂.
 16. The methodaccording to claim 3, wherein the calcium-containing solution furthercomprises Mg.
 17. The method according to claim 3, wherein thecalcium-containing solution further comprises MgCl₂.
 18. The methodaccording to claim 17, wherein the calcium-containing solution furthercomprises CO₃ ²⁻ counterion.
 19. The method according to claim 17,wherein said method further comprises adding CO₃ ²⁻ counterion to thecalcium-containing solution.
 20. The method according to claim 19,wherein the CO₃ ²⁻ counterion is added to the calcium-containingsolution by peristaltic pumping.
 21. A method for sequestering alipophilic agent within a patient comprising administering an effectiveamount of core-shell particles to the patient, wherein the core-shellparticles comprise: (a) a shell, wherein the shell comprises calciumcarbonate; (b) a core; and (c) an intermediate layer between the shelland the core, wherein the intermediate layer comprises an amphiphiliccompound, and wherein the core and the intermediate layer are surroundedby the shell.
 22. The method according to claim 21, wherein the core ishollow.
 23. The method according to claim 21, wherein the core comprisesan oil.
 24. The method according to claim 23, wherein the core furthercomprises an enzyme that degrades the lipophilic agent.
 25. The methodaccording to claim 21, wherein the enzyme comprises a cytochrome P450enzyme.
 26. The method according to claim 24, wherein the lipophilicagent is absorbed into the core-shell particles, and wherein the enzymesubsequently degrades the lipophilic agent.
 27. The method according toclaim 21, wherein the core-shell particles are administered to thepatient intravenously.
 28. The method according to claim 21, wherein atoxic amount of the lipophilic agent is present within the patient priorto said administration of the core-shell particles.
 29. The methodaccording to claim 24, wherein the enzyme is adsorbed onto the shell ofthe particles.
 30. A method for sequestering a lipophilic agent from thesurrounding environment comprising contacting an effective amount ofcore-shell particles with the lipophilic agent, wherein the core-shellparticles comprise: (a) a shell, wherein the shell comprises calciumcarbonate; (b) a core; and (c) an intermediate layer between the shelland the core, wherein the intermediate layer comprises an amphiphiliccompound, and wherein the core and the intermediate layer are surroundedby the shell.
 31. The method according to claim 30, wherein saidcontacting is carried out in vivo.
 32. The method according to claim 30,wherein the surrounding environment is a patient's bloodstream.
 33. Amethod for delivering a biologically active agent to a patientcomprising administering an effective amount of core-shell particles tothe patient, wherein the core-shell particles comprise: (a) a shell,wherein the shell comprises calcium carbonate; (b) a core, wherein saidcore comprises a biologically active agent; and (c) an intermediatelayer between the shell and said core, wherein said intermediate layercomprises an amphiphilic compound, and wherein said core and saidintermediate layer are surrounded by said shell.